Gaucher disease is the name given to a group of lysosomal storage disorders caused by mutations in the gene that codes for an enzyme called glucocerebrosidase ("GC"). Gaucher disease is caused by deficiency of GC as reported by Patrick, A. D., Biochem. J. 97:17C (1965) and Brady, R. O., et al., Biochem. Biophys. Res. Commun. 18:221 (1965). All of the mutations in the gene alter the structure and function of the enzyme which lead to an accumulation of the undegraded glycolipid substrate glucosylceramide, also called glucocerebroside, in cells of the reticuloendothelial system. Each particular mutation of the human GC gene leads to a clinical disease collectively known as Gaucher disease. These disorders are usually classified into three types; type 1 (non-neuronopathic), type 2 (acute neuronopathic) and type 3 (subacute neuronopathic), the type depending on the presence and severity of neurologic involvement. Gaucher disease is the most prevalent Jewish genetic disease and the most common lysosomal storage disease.
GC is a monomeric, membrane-associated, hydrophobic glycoprotein with a molecular weight of 65,000 daltons. Human GC contains 497 amino acids and is translated as a precursor protein with a 19 amino acid hydrophobic signal peptide which directs its co-translational insertion into the lumen of the endoplasmic reticulum-golgi-lysosome complex as reported by Erickson, A. H., et al., J. Biol. Chem. 260: 14319 (1985). GC acts at the acidic pH of the lysosome to hydrolyze beta-glucosidic linkages in complex lipids ubiquitously found in all membranes to form the byproducts of glucose and ceramide. The catalytic activity of GC is increased in vitro by detergents, lipids, and in vivo by a naturally occurring activator known as sphingolipid activator protein-2 (SAP-2 or saposin C). See, Ho, M. W., et al., Proc. Natl. Acad. Sci. USA 68:2810 (1971); and O'Brian, J. S., et al., Science 241:1098 (1988).
Human GC cDNA was first cloned as described by Ginns, E. I., et al., Biochem. Biophys. Res. Commun. 123:574 (1984). Subsequent characterizations of other GC cDNA clones by, for example, Sorge, J., et al., Proc. Nat. Acad. Sci. USA 82:7289 (1985) and Tsuji, S., et al., J. Biol. Chem. 261:50 (1986), have led to the elucidation of the complete nucleotide sequence of human GC. As reported by Ginns, E. I., et al., Proc. Nat. Acad. Sci. USA 82:7101 (1985), the GC gene was localized to human chromosome lq21 by in situ hybridization. Tsuji, S., et al., New Enql. J. Med. 316:570 (1987), have shown that the GC gene comprises 11 exons and 10 introns spanning approximately 7 Kb.
While more than twenty mutations in the human GC gene are known, only two are common. See, Tsuji, S., et al., Proc. Natl. Acad. Sci. USA 85:2349 (1988). The two common mutations account for approximately 70% of the mutant alleles, as reported by Firon, N., et al., Am. J. Hum. Genet. 46:527 (1990). Mutant GC genes code for aberrant proteins that are either catalytically altered or unstable and rapidly disappear from the cell.
Although GC is deficient in all of a subject's cells, for unknown reasons, the accumulation of the substrate glucosylceramide occurs virtually only in macrophages. Gaucher disease is unique among lysosomal storage disorders for this reason, that is, causing storage within only one cell type. This characteristic of the pathobiology of the disease has led to the development of two successful treatment strategies based on correcting the enzyme deficiency in macrophages.
The first of the two Gaucher disease treatments based on this strategy is allogeneic bone marrow transplantation, which results in the repopulation of affected tissues with enzyme-competent macrophages. See, Rappeport, J. M., et al., Birth Defects: Original Article Series 22,1:101 (1986). The second approach to treatment which has resulted in clinical improvement in Gaucher disease patients is macrophage-targeted enzyme replacement. This treatment takes advantage of naturally occurring mannose receptors on macrophages and the exposition of accessible mannose receptors in the oligosaccharides of glucocerebrosidase to efficiently deliver the enzyme to macrophages. See, Barranger, J. A., et al., Japanese J. of Inher. Met. Disease 51:45 (1989); Takasaki, S., et al., J. Biol. Chem. 259:10112 (1984); and Furbish, F. S., et al., Biochem. Biophys. Acta. 673:425 (1981). While both of these approaches to treating Gaucher disease are important because they provide some means of therapy where none previously existed, both approaches have significant limitations. Allogeneic bone marrow transplantation has associated with it morbidity and mortality risks that are unacceptable for many patients. Further, HLA matched bone marrow donors do not exist for the majority of patients. As for macrophage-targeted enzyme replacement, it is currently an expensive and life-long therapy; thus, it should be reserved for only the most severely ill patients.
Despite the limitations of these two therapies, their successes have demonstrated that enzymatic correction of only one cell type, the macrophage, results in effective therapy for Gaucher disease. From the point of view of developing somatic cell gene therapy for Gaucher disease, the fact that marrow transplantation is effective demonstrates that bone marrow stem cells are an appropriate target cell to which to transfer a "therapeutic" gene. Further, because of the pivotal role of macrophages in Gaucher disease, alternative target cells to be considered for gene transfer are the committed macrophage precursors, peripheral blood monocytes, or cultures of bone marrow which are capable of producing macrophage precursors.
To be an effective permanent treatment for any disease capable of being treated by gene therapy, the transfer and sustained expression of genes in cells important to the pathogenesis of the particular disease is required. Sufficient expression of a transduced GC gene in the progeny of pluripotent bone marrow stem cells would likely correct the deficiency of the enzyme in all cell series including monocytes/macrophages. Experience from allogeneic marrow transplantation and macrophage-targeted enzyme replacement supports the idea that gene therapy would provide a cure provided adequate expression of the GC gene were achieved in a sufficient number of macrophages.
Much experience has been gained recently to evaluate the efficiency of gene transfer and expression in bone marrow stem cells using replication defective retroviral vectors. See, e.g., Miller, A. D., Blood 76:2 (1990) and Miller, D. G., et al., Mol. Coll. Biol. 10:4239 (1990). Most of the studies of retroviral vectors have been conducted in the mouse model of bone marrow transplantation. Recent data show that 10-20% of stem cells can be transduced and survive to repopulate marrow. See, Bodine, D. M., et al., Exp. Hematol. 19:206 (1991). Many fewer studies have been conducted in larger animals and, at the present time, experimental conditions have not yet been fully optimized. For the mouse model, critical parameters for efficient retroviral gene transfer and repopulation of bone marrow include high titer virus producer cell lines (VPL), pretreatment of mice with 5-fluorouracil (5-FU) to initiate stem cell cycling, pre-culture of bone marrow with growth factors including IL-3 and IL-6 and stem cell factor (SCF). Several studies have shown that the GC gene can be transferred to murine bone marrow stem cells and their progeny, but until recently none had demonstrated expression of enzymatic activity in macrophages in vivo. See, Nolta, J. A., et al., Blood 75:75 (1990) and Correll, P. H., et al., Proc. Natl. Acad. Sci. USA 86:8912 (1989).
Retroviral vectors for use in gene therapy require dividing cells in order to integrate and they have a small, but finite chance of interrupting an essential gene or altering expression of a gene proximate to the site of integration of the retroviral provirus. However, the proviral integration may be preferentially directed to transcriptionally active regions of the genome as described by Scherdin, U., et al., J. Virol. 64; 2:907 (1990). These requirements of retroviral vectors are believed to contribute to the small number of stem cells that can be transduced, since only a portion of the stem cell population is cycling even under optimal experimental conditions. See, McLaughlin, S. K., et al., J. Virol. 65:1963 (1991).
For these and other reasons, the small helper-dependent human DNA parvovirus known as adeno-associated virus ("AAV") has recently received attention as a vector that could be useful for gene therapy. See, Hunter, L. A., et al., J. Virol. 66:317 (1992); Tratschin, J. D., et al., Mol. Cell. Biol. 5:3251 (1985); Hermanat, P. L., et al., Proc. Natl. Acad. Sci. USA 81:6466 (1984); and Lebkaski, J. S., et al., Mol. Cell. Biol. 8:3988 (1988). A lytic growth cycle for wild AAV requires infection with a helper virus, e.g., adenovirus type 5. In the absence of helper virus, AAV integrates into the host genome by hybridization between the AAV terminal repeats (TR) and host sequences in a stable manner thereby establishing a permanent latent infection. In human cells, this integration occurs preferentially in a single silent site in human chromosome 19. See, Samulski, R. J., et al., EMBO J. 10:3941 (1991); Kotkin, R. M., et al., Proc. Natl. Acad. Sci. USA 87:2211 (1990).
In order to develop gene therapy that would be useful for the treatment of Gaucher disease as well as other hematopoietic disorders, new vectors are needed that allow efficient gene transfer to stem cells and at the same time direct expression of the transferred gene. Thus far there has not been a vector that can transduce and sustain the expression of the human GC gene in bone marrow stem cells and their progeny. Accordingly there is a need for such a vector which could be used in gene therapy for Gaucher disease.